Caenorhabditis elegans brc-1 mutation increases the number of COSA-1 foci in him-8 and zim-2 mutants

Crossover designation factors such as COSA-1 are concentrated at the specific DNA double-strand break (DSB) sites to promote crossover formation. zim-1 mutants, which show defects in the homologous chromosome pairing of chromosomes II and III, increase the COSA-1 foci/normal bivalent state compared to the expected value. The excess designation was suppressed by an additional mutation in brc-1 in zim-1 mutants. We demonstrated that the number of COSA-1 foci in him-8 and zim-2 mutants, showing defects in the pairing of the X and V chromosomes, respectively, increased compared to the expected value, and brc-1 mutation accelerated the number of COSA-1 foci in oogenesis.


Description
Meiosis is a cell division process that generates haploid gametes from diploid parental cells.Crossover formation is essential for appropriate homologous chromosome segregation of meiosis I.A single crossover event per homologous chromosome pair is observed in wild-type Caenorhabditis elegans.The process of selecting a specific DNA double-strand break (DSB) as a future crossover site is called crossover designation (Kleckner et al. 2003;Gray and Cohen 2016).Crossover designation factors such as COSA-1, MSH-5, and ZHP-3 concentrate at the sites of DSBs, which are further repaired by crossover (Kelly et al. 2000;Jantsch et al. 2004;Bhalla et al. 2008;Yokoo et al. 2012).Therefore, six COSA-1 foci are observed in the nucleus at the late pachytene stage in the wild-type (Yokoo et al. 2012).
We quantified the GFP::COSA-1 foci in the late pachytene stage in the wild-type, him-8, brc-1, zim-2 single mutants, and brc-1; him-8, brc-1; zim-2 double mutants by 3D-fluorescent microscopy.As previously shown by many groups, six GFP::COSA-1 foci were observed in the wild-type (Figure 1A, 1B).If a single crossover event was hypothesized to occur per homologous chromosome pair, then we would expect to observe five GFP::COSA-1 foci/nucleus in him-8 mutants with unpaired X chromosomes and in zim-2 mutants with unpaired chromosome V.However, in addition to five foci of GFP::COSA-1/nucleus, we observed that 30.5% and 65.4% of cells had more than six foci of GFP::COSA-1 in him-8 and zim-2 mutants, respectively (Figure 1A, 1B).This phenotype was similar to that observed in zim-1 mutants (Li et al. 2018).These data suggest that the interchromosomal effect causes excess crossover in a normal pair of homologous chromosomes if crossover formation does not occur in one or two sets of unpaired chromosomes during C. elegans oocyte meiosis (Herman and Kari 1989;Carlton et al. 2006;Li et al. 2018).A similar increase in crossover formation between normal chromosome pairs has been observed during Drosophila female meiosis when chromosomal rearrangements (heterozygous inversions or translocations) are present in the cell (Sturtevant 1919;Sturtevant 1921).
One of the major roles of BRC-1 is to stabilize RAD-51 filaments when crossover formation is impaired (Janisiw et al. 2018;Li et al. 2018).RAD-51 levels in pachytene are elevated in him-8 and zim-1 mutants compared to those in the wild-type (Carlton et al. 2006;Li et al. 2018).Removal of BRC-1 in zim-1 mutants results in a "dark zone" of RAD-51 in mid to late pachytene in which the accumulation of RAD-51 foci are suppressed (Li et al. 2018).To examine whether BRC-1 promotes RAD-51 filament stability in him-8 mutants, we compared RAD-51 levels in four individual zones from the first cell of the transition zone to the end of late pachytene in the wild-type, him-8, and brc-1; him-8 double mutants (Figure 1C, 1D, 1E).Similar to a previous study, the levels of RAD-51 foci were elevated in all pachytene stages (zones 2, 3, and 4) in him-8 mutants compared with the wild-type (Carlton et al. 2006) (Figure 1C, 1E).The RAD-51 levels were decreased to 81% in mid-pachytene (zone 3) in brc-1; him-8 double mutants compared to him-8 single mutants (p<0.0001); however, obvious RAD-51 dark zone was not observed in brc-1; him-8 double mutants.Most RAD-51 foci remain in the late pachytene stage (zone 4) in brc-1; him-8 double mutants compared with him-8 single mutants (p<0.0001).These results suggest that the contribution of BRC-1 to RAD-51 filament stability at mid-pachytene in him-8 mutants was not as high as that in the zim-1 mutant.
We observed an excess number of GFP::COSA-1 foci in the him-8 and zim-2 single mutants compared with the expected value of the five autosome pairs.These findings are consistent with those of previous studies, which demonstrated that crossover formation increases in autosomes during oogenesis in him-8 mutants (Herman and Kari 1989;Carlton et al. 2006).These data suggest that interchromosomal effects occur in him-8 and zim-2 mutants, similar to zim-1 mutants (Li et al. 2018).
The phenotypes of brc-1; him-8 (zim-2) and brc-1; zim-1 differed during oogenesis.The brc-1 mutation suppresses the formation of COSA-1 foci in zim-1 mutants (Li et al. 2018), whereas the brc-1 mutation enhanced the formation of COSA-1 foci in him-8 and zim-2 mutants in the present study.A RAD-51 dark zone was observed in brc-1; zim-1 double mutants (Li et al. 2018), whereas in brc-1; him-8 double mutants, a slight decrease in RAD-51 levels, but no clear RAD-51 dark zone, was observed.These observations suggest that the function of BRC-1 in the formation of COSA-1 foci and the processing (removal/stabilization) of RAD-51 may be regulated differently according to the number of chromosome pairs involved in oogenesis.Furthermore, COSA-1 foci formation is differentially regulated during oogenesis and male spermatogenesis (Li et al. 2020).The single X chromosome condition in wild-type, him-8 mutant, and brc-1; him-8 double mutant males did not increase the number of COSA-1 foci compared with the hypothesized number of five COSA-1 foci (Li et al. 2020).In contrast to oogenesis, brc-1; zim-1 males demonstrated enhanced formation of COSA-1 foci compared to zim-1 single mutants (Li et al. 2020).Further research is required to explore the sex-specific regulation of crossover designs under pairing defect conditions.

Fixation and immunostaining:
Worms expressing GFP::COSA-1 (post L4 22-24 h) were dissected using a scalpel.For dissection, 30 µL of 15 mM sodium azide solution was placed on a cover glass, followed by approximately 20 worms.After removing 15 µL of the sodium azide solution, 15 µL of 2% Paraformaldehyde (PFA) was added, mixed to a final concentration of 1% PFA and left for 5 min to fix.After removing 15 µL of the mixture, the sample was sandwiched in a slide glass.The slide was placed at -80°C for 5 min.After removing the cover glass by cracking it with a razor blade, it was fixed again in ice-cold methanol (-20°C) for 1 min.Immunostaining was performed as previously described (Saito et al. 2009).The primary and secondary antibodies used in this study were rabbit anti-RAD-51 antibody ( (Das et al. 2022), 1:3,000) and goat FITC-conjugated anti-rabbit antibody (Jackson ImmunoResearch, 1:200), respectively.The sample was then washed with phosphate-buffered saline with Tween 20 for 5 min for three times, mounted with 8 µL VECTASHIELD with 4',6-diamidino-2-phenylindole (Vector laboratories, Burlingame, California, USA), and the cover glass was shielded by nail polish.